DNA gyrase is an essential type II topoisomerase that catalyzes the introduction of negative supercoils using the free energy of ATP hydrolysis. The enzyme is composed of two subunits, Gyrase A (GyrA) and Gyrase B (GyrB), that form a functional heterotetramer A2B2 required for bacterial viability. The GyrA subunit is targeted by synthetically developed quinolone antibiotics, which have broad-spectrum activity against both gram-positive and gram-negative bacteria. The GyrB subunit is targeted primarily by natural product antibiotics such as the aminocoumarin antibiotics, e.g., novobiocin and coumermycin1, as well as cyclothialidine2. Mutations that confer drug resistance to all three antibiotics have been reported3; 4. Mutations associated with both coumarin and cyclo-thialidine resistance map to the periphery of the ATP binding site of GyrB that hydrolyzes ATP5. The emergence of bacterial strains resistant to existing antibiotics makes it imperative to develop new classes of antibiotics that take into account these known mutations and, to the extent possible, restrict their mode of action to portions of the enzyme that are conserved by functional necessity. Residues required for coupling ATP hydrolysis to DNA supercoiling in GyrB have been identified using site-directed mutagenesis5. Along with this extensive mutational data, analysis of high-quality crystal structures suggests the value of pursuing next-generation GyrB inhibitors that target the ATP-binding domain6. The ATP binding site within GyrB is highly conserved across bacterial species and is not present in humans, making it suitable for the development of broad-spectrum antibiotics.
Because the bacterial gyrase holoenzyme has been the subject of multiple drug discovery efforts, many assays exist to measure its activity. Assays used for general studies of the holoenzyme as well as many high-throughput screens measure the ability of the enzyme to convert relaxed DNA into supercoiled DNA. Most of these studies use an assay that couples ATP hydrolysis to NADH, resulting in a measureable colorimetric change2; 5; 6. Similar assays directly measure the total level of supercoiled DNA using agarose-gel separations or fluorescence dyes7-9. Cell-based assays that measure the level of DNA damage have also been used to measure gyrase activity10. Although all of these assays can be used to measure the activity of the gyrase holoenzyme, they often require multiple addition steps, cannot separate GyrA from GyrB inhibitors, and do not focus on the ATP-binding domain. One assay has been described that measures the direct binding of [3H]dihydronovobiocin to a biotin-labeled 43-kDa fragment of GyrB using a scintillation proximity assay (SPA)10. While the SPA directly examines the ATP-binding domain, an assay that does not require radioactivity would be more suitable for high-throughput screening (HTS).
Fluorescence polarization (FP) is a homogeneous assay that can be used to measure the binding interaction between two molecules11. FP is based on the principle that a fluorophore excited by polarized light will also emit polarized light. Molecular motion, which is dependent on the size of the molecule, causes depolarization of the light by radiating at a different direction than the incident light. A small unbound fluorescent probe rotates rapidly and maintains low levels of polarization after excitation. If the fluorescent probe binds to a larger molecule, such as a protein, forming a stable complex, the bound probe rotates more slowly and increases the amount of polarized light. Binding is directly related to the polarization level of the sample: an unbound fluorescent probe has low FP and a bound fluorescent probe has high FP. The FP assay is well suited for measuring the interaction of two molecules in real time and is commonly used in HTS12.
This patent presents the development and optimization of a novel FP assay to detect competitive inhibitors of the ATP-binding domain of GyrB and structurally-related topoisomerases. We have designed and synthesized a novel fluorescent probes by covalently attaching a fluorophores to novobiocin guided by the GyrB/novobiocin crystal structure (Protein Data Bank entry 1KIJ)13. Experiments were performed to develop the FP assay and optimize the use of the conjugates to measure the competition for binding to the ATP-binding domain of GyrB. We have determined the kinetics and strength of the interaction of the conjugates with GyrB as well as the effect of common buffer additives on the interaction. The assay was also validated for use in HTS for inhibitors of the ATP-binding domain by screening a small library of FDA-approved compounds. This screen identified a known GyrB inhibitor as well as four members of the anthracycline family of cancer therapeutics (doxorubicin, idarubicin, epirubicin, and daunorubicin).
The subject probe conjugates were nonobvious and their use in FP assays was unexpected. Labeling small-molecule ligands with fluorescent dyes is uncommon for FP assays, since the labeling chemistry often results in substantial increases in molecular weight and alterations of molecular properties. Our design strategy was to utilize a natural product aminocoumarin that tightly binds into the ATP binding site of the topoisomerase, and tether a fluorescent dye off of a solvent-exposed position of the molecule. We studied the crystal structure of novobiocin bound to GyrB and determined that most of the ligand is deeply buried within the protein active site, with the exception of the phenolic benzamide ring, so our proposed modification was modeled in the active site to confirm solvent accessibility of the attached moiety. Since the phenol group was directly interacting with an aspartic acid residue, we hypothesized that the ortho position of this ring would be a good position at which to attach a fluorescent labeling group. We specifically labeled this position of novobiocin using a Mannich reaction, which is selective for aromatic ring positions ortho to a phenol group, and under neutral conditions, as we knew that novobiocin degrades under either basic or acidic conditions.
Topoisomerase IV is a bacterial type II topoisomerase of similar structure and function: it also utilizes the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome, has the same subunit structure—wherein the subunits corresponding to the gyrase subunits A and B are named C and E (Bellon et al. Antimicrob Agents Chemother 2004, May, 48(5), 1856-64) and is similarly inhibited by aminocoumarins and targeted by the subject probes.
Aspects of this disclosure were published by us in: Glaser et al., J Biomol Screen. 2011 February; 16(2):230-8. Epub 2011 Jan. 18, entitled “A high-throughput fluorescence polarization assay for inhibitors of gyrase B.”